International Biodeterioration & Biodegradation 107 (2016) 140e146

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International Biodeterioration & Biodegradation

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Phenol biodegradation by halophilic archaea

Eda Acikgoz a, Birgul Ozcan b, *
a
Ege University, Faculty of Medicine, Histology & Embryology Department, 35100 Izmir, Turkey
b
Mustafa Kemal University, Sciences and Letters Faculty, Biology Department, 31024, Hatay, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Phenol is a toxic aromatic compound produced as a by-product of industrial activities. Biological
Received 13 July 2015 treatment of highly saline wastewaters containing phenol can be performed through halophilic micro-
Received in revised form organisms. In this study, the ability of halophilic archaeal isolates to degrade phenol was investigated.
16 November 2015
Among 103 tested isolates, the strain designated A235 was identified as having the highest phenol
Accepted 18 November 2015
Available online xxx
degradation capacity on solid and liquid media containing 20% (w/v) NaCl and phenol as the sole carbon
and energy source. The strain was adapted sequentially to increasing phenol concentrations. The removal
of phenol via cross-toluene adaptation was increased by 14% in the medium. The growth kinetics of strain
Keywords:
Halophilic archaea
A235 during growth on phenol was found to fit the Monod model. The values of mmax and Ks were
Phenol biodegradation calculated to be 0.015 h
1 and 71.4 g l
1, respectively. For an initial phenol concentration of 100 ppm, the
Adaptation biodegradation by A235 was found to be optimal at pH 7.5, 37  C and 200 rpm when the culture con-
Catechol 2,3-dioxygenase tained 20% (w/v) NaCl, 0.025% yeast extract and the inoculum size was set at 10%. A preliminary enzyme
screening indicated that the degradation of phenol was achieved through a meta-cleavage pathway
involving a catechol 2,3-dioxygenase. Catechol 2,3-dioxygenase displayed its highest catalytic activity at
42  C, 2 M KCl, and pH 8. To the best of our knowledge, this is the first report showing the ability an
extremely halophilic archaeon to metabolize phenol at higher salt concentrations.
© 2015 Published by Elsevier Ltd.

1. Introduction concentrations (from 3 to 35%) (Ventosa et al., 1998; Bastos et al.,

Phenolic compounds derived from industrial activities are
ubiquitous in the environment. Since they are toxic to many or-
ganisms, they must be removed. It is estimated that 5% of industrial
effluents are saline or hypersaline (Borgne et al., 2008). Phenol
removal from hypersaline wastewaters is frequently achieved by
physicoechemical approaches such as precipitation, coagulation,
ion exchange, ultra-filtration or evaporation. These processes are
usually energy-consuming and lead to secondary effluent prob-
lems. As an alternative, the implementation of biological processes
increasingly gains interest, especially in the case of phenol since
microorganisms can use it as sole source of carbon and energy. On
the other hand, saline conditions may limit microbial degradation
of phenol and make conventional biodegradation processes inef-
fective. Conventional microorganisms do not survive under these
saline conditions. However, halophilic microorganisms have
adapted to grow optimally in a very wide range of salt
* Corresponding author.
E-mail addresses: acikgozedaa@gmail.com (E. Acikgoz), birgulozcan@gmail.com
(B. Ozcan).
2000; Margesin and Schinner, 2001).
These halophiles are good candidates for the bioremediation of
hypersaline environments including saline effluents. The ability of
several halophilic and halotolerant bacteria to biodegrade phenol
has been demonstrated (Bastos et al., 2000; Alva and Peyton, 2003).
For instance, biodegradation of phenol in hypersaline wastewaters
was achieved by employing moderate halophiles (up to 15% salt) as
reported by Woolard and Irvine (1994). Haddadi and Shavandi
(2013) reported a Halomonas sp. strain that exhibited the ability
to grow on phenol as the sole source for carbon and energy in the
presence of 18% (w/v) NaCl. Although the biodegradation of phenol
by halophilic and halotolerant bacteria has been well demon-
strated, the information on phenol biodegradation by halophilic
archaea has not been recorded yet. On the other hand, the capacity
of halophilic archaea to degrade some other organic substrates (p-
hydroxybenzoic acid, naphthalene, phenanthrene, and pyrene) has
been documented (Erdogmus et al., 2013).
Since phenols are toxic to many microorganisms, different ap-
proaches, such as adapting the cells to higher phenol concentra-
tions, immobilizing the microbial cells or adding a co-substrate
such as yeast extract, glucose, carbohydrates and fatty acids have

http://dx.doi.org/10.1016/j.ibiod.2015.11.016
0964-8305/© 2015 Published by Elsevier Ltd.
 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146
been suggested to overcome the phenol toxicity (Masque et al.,
1987; Lob and Tar, 2000). Among these approaches, adaptation to
higher phenol concentrations is the most effective to overcome
substrate inhibition (Masque et al., 1987). In addition, pre-adaptation
to phenol may have significant effects on the degradation patterns
such as shorter degradation time or higher degradation rates
(Kwon and Yeom, 2009). The mechanism of microbial adaptation to
phenol degradation was explained on the basis intracellular
enzyme induction (Yeom et al., 1997). Microbial degradation of
phenol under aerobic conditions usually proceeds via an initial
hydroxylation to generate catechol which is cleaved through the
ortho or meta fission pathways (Bonfa et al., 2013). The ortho
cleavage catalysed by the catechol 1,2-dioxygenase (EC 1.13.11.1)
leads to the formation of cis,cis-muconic acid and the meta-cleavage
catalyzed by the catechol 2,3-dioxygenase (EC 1.11.13.2) leads to the
formation of 2-hydroxymuconic semialdehyde (Gurujeyalakshmi
and Oriel, 1989).
In this work, we have investigated the ability of the halophilic
archaeon strain A235 to use phenol a sole carbon source at high salt
concentrations and we have obtained preliminary evidence that in
this strain, phenol degradation proceeds through the meta-cleavage
pathway. We also determined its growth kinetics during the
degradation of phenol in batch culture. The effects of phenol and
toluene adaptation as well as the effects of various growth condi-
tions on phenol degradation were also investigated.
2. Materials and methods
2.1. Screening for phenol-degrading archaeal strains
A total of 103 halophilic Archaea isolated from different parts of
Turkey were routinely cultured in SehgaleGibbons (SG) medium as
explained before (Ozcan et al., 2006). The agar plates contained 2%
(w/v) agar. The liquid cultures were incubated at 120 rpm and at
37  C.
The halophilic archaeal isolates were screened for their ability to
degrade phenol at 37  C on a solid or liquid mineral salt medium
containing phenol (PMS). The PMS medium was modified from
Halohandbook online protocol (Dyall-Smith, 2009). The medium
contained (g l
1): NaCl 200; MgSO4.7H2O 25; MgCl2.6H2O 10;
CaCl2.2H2O 1.25; KCl 5; K2HPO4 8.7; KH2PO4 6.8; trisma baze 6;
NH4Cl 0.2; phenol and trace element 2 ml l
1 containing ((g/
100 ml) MnCl2.4H2O 0.03; ZnSO4.7H2O 0.1; FeCl3 0.25; CuSO4.5H2O
0.01; Na2MoO4.2H2O 0.03; H3BO4 0.3; NH4NO3 0.2). The pH of the
medium was adjusted to 7.35. Preliminary screening was carried
out by inoculating archaeal cell suspensions on PMS-agar plates
containing 50 ppm (0.5 mM) phenol as sole carbon source. Cells
capable of degrading phenol were successively transferred to an
identical medium containing increasing concentrations of phenol
ranging from 100 to 200 ppm. The highest phenol tolerant strains
were inoculated in liquid PSM containing 100, 150, 200 and
250 ppm (1.1, 1.6, 2.1 and 2.7 mM, respectively) phenol for quan-
titative screening. The cultures were incubated at 120 rpm. The
most efficient phenol degrading strain (strain A235) was selected
by monitoring the optical density (OD600) of the cultures after 15
days of incubation. Cells employed for phenol degradation were
obtained from the late exponential phase cultures (OD600~3) by
centrifugation at 5000 rpm for 30 min at 4  C and washing twice
with 50 mM TriseHCl buffer (pH 7.35). All cultures were incubated
at 37  C.
2.2. Analysis of archaeal growth and phenol degradation
Archaeal growth was carried out with various initial phenol
concentrations ranging from 50 to 200 mg l
1. Cell concentration
141
was calculated by plotting the colony forming units (CFUs) of the
culture vs. optical density measured at 600 nm.
Phenol concentration was determined spectrophotometrically
at 500 nm by monitoring the production of 4-aminoantipyrine
(Yang and Humphrey, 1975). This method is based on rapid
condensation with 4-aminoantipyrine followed by oxidation with
alkaline potassium ferricyanide. All samples were analysed three
times to calculate an average value. The specific growth rates for
various initial phenol concentrations were calculated from the
growth kinetic slope.
2.3. Adaptation to phenol and toluene
Adaptation of isolate A235 to phenol was achieved by gradually
increasing the phenol concentration of the medium, from 50 to
250 ppm and simultaneously decreasing the yeast extract con-
centration from 0.075 to 0%. Similarly, toluene adaptation was
achieved by increasing the concentration of toluene. The initial
toluene concentration was set at 10 ppm and it was changed
stepwise to 20, 30, 40 and 50 ppm. The adapted cells were then
tested for their phenol degradation capacities.
2.4. Effects of growth conditions on phenol degradation
The optimal cultural conditions for phenol biodegradation by
strain A235 were determined by using different concentrations of
phenol (50, 75, 100, 150 and 200 mg l
1), NaCl (10, 15, 20 and 25 M)
and yeast extract (0.3, 0.1, 0.005, 0.025%), and different inoculum
sizes (2.5, 5,10, 15% v/v), pH (6, 7, 7.5, 8 and 8.5), temperatures (25,
30, 37, 45 and 50  C) and agitation rates (120, 140, 170 and
200 rpm). For each variable, optical density and phenol concen-
trations were monitored during the cultivation.
2.5. Rothera test for the detection of ortho- and meta-cleavage
products
The ring cleavage pattern of catechol by isolate A235 was
determined by the Rothera's test (Ottow and Zolg, 1974). The test
was performed as follows: the cell pellet was suspended in 2 ml
0.02 M Tris buffer (pH 8), then 0.5 ml of toluene and 0.2 ml of 4 mM
catechol were added. Following vigorous shaking of the tube, the
development of a yellow colour within 5 min was considered as an
indication of meta-cleavage. In the absence of yellow colour,
ammonium sulphate (1 g) was added and the mixture was further
incubated at 30  C for 1h. The addition of freshly prepared sodium
nitroprusside and 2.0 ml of concentrated ammonium hydroxide
develops a strong violet colour when the degradation proceeds
through the ortho-clevage pathway.
2.6. Enzyme assays
2.6.1. Preparation of cell extracts
Cells grown to exponential phase under optimal degradation
conditions were harvested by centrifugation at 15,000 rpm for
15 min at 4  C and washed twice with 50 mM TriseHCl buffer (pH
7.35). The pellet was suspended in the same buffer containing KCl
and the cells were disrupted by sonication (Bandelin Sonopuls GM
200) on ice for a total time of 6 min (30 s on/30 s off). Cell debris
was removed by centrifugation at 15,000 rpm for 30 min at 4  C and
the supernatant was used to assay the enzymes activities.
2.6.2. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase
activities
Catechol 1,2-dioxygenase (EC 1.14.13.1) and catechol 2,3-
dioxygenase (EC 1.13.11.2) activities were measured
142 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146
spectrophotometrically. All assays were performed at room tem-
perature and one unit of activity was defined as the amount of
enzyme transforming 1 mmol of substrate in 1 min under the assay
conditions. Catechol 1,2-dioxygenase was assayed by measuring
the absorbance at 260 nm, corresponding to the formation of cis,cis-
muconic acid (Hegeman, 1966). The reaction mixture contained
0.1 ml of crude extract, 50 mM TriseHCl buffer (pH 7.5) and 3.3 mM
2-mercaptoethanol. Following the addition of catechol, the activity
was monitored for 5 min. The activity of catechol 2,3-dioxygenase
was measured as previously described (Feist and Hegeman, 1969),
from the increase of absorbance at 375 nm, corresponding to the
production of 2-hydroxymuconic semialdehyde, the meta-cleavage
product of catechol. The reaction mixture was composed of crude
extract (0.2 ml), 50 mM TriseHCl buffer (pH 7.5) and catechol
(0.2 ml). The reaction was incubated for 5 min.
2.6.3. The effects of salt concentrations, temperatures and pH on
catechol 2,3-dioxygenase activity
The effect of KCl concentrations (1.5e3.5 M) on the enzyme
activity was determined at 25  C in 50 mM Tris buffer (pH 7.5). The
effects of temperatures and pH were determined at the optimal KCl
concentration by carrying out reactions at various temperatures
(from 20 to 52  C) and pH (from 5 to 8.5).
In the present study, all tests were performed in triplicate.
3. Results and discussion
3.1. Screening of phenol degrading strains
In this study, 103 archaeal strains were screened on solid PMS
containing between 50 and 200 ppm phenol as sole carbon source.
Among the 103 isolates, 8 were able to tolerate 200 ppm phenol.
These eight isolates were grown in liquid PMS containing 50 ppm
phenol. After 20 days incubation, the tested isolates were able to
degrade between 5 and 42% phenol. Three isolates (A235, A405 and
A138) were selected as they displayed phenol degradation above
20%. These isolates were inoculated in liquid PMS containing 100,
150 and 200 ppm phenol. Isolate A235 was finally retained for
further work, as it was the most effective phenol-degrading strain
(reaching 32% degradation at 150 ppm phenol). There was no
growth in the medium containing 200 ppm phenol.
In our previous studies, it was demonstrated that 95 of the 103
isolates contained the phytanil diether moieties which is a typical
feature of Archaea (Ozcan et al., 2006). In addition, 45 of them were
subjected to 16S rRNA gene sequencing analyses (Yildiz et al., 2012;
Ozcan et al., 2012, 2007) which revealed that they were members of
Halobacteriaceae family. We have not determined the precise
taxonomic position of strain A235 which was the best phenol
degrading strain. However, according to our previous findings
(Ozcan et al., 2006), this strain was found to bear several features
common to archaea and on the basis of its protein profile, it clus-
tered with the archaea isolate designated A283. It was clearly
shown that the isolate A283 belongs to the genus Haloarcula from
the domain Archaea on the basis of its 16S rDNA gene sequence and
of its polar lipid profile (Ozcan et al., 2007).
Moderately halophilic or salt tolerant microorganisms grown at
up to 14% (w/v) NaCl were reported to use different phenol con-
centrations up to 1600 mg l
1 (Hinteregger and Streichsbier, 1997;
Bastos et al., 2000; Munoz et al., 2001; Alva and Peyton, 2003;
Maskow and Kleinsteuber, 2004; Bonfa et al., 2013). Hong-Xia
(2011) reported that the bacterial strain SM5 which was isolated
from a marine environment degraded more than 93% phenol at
5e25 g l
1 NaCl. Although there are a few studies showing the
ability of halophilic archaea to use aromatic substrates (e.g. p-
hydroxybenzoic acid, benzoic acid, cinnamate, phenylpropanoate)
as sole carbon and energy sources (Emerson et al., 1994; Fairley
et al., 2002; Cuadros-Orellanaa et al., 2012), we are not aware of
any previous study showing the ability of members of this group to
degrade phenol.
3.2. Adaptation to phenol and toluene
During the acclimatization process, the microorganisms develop
strategies to overcome the substrate inhibition problems (Lob and
Tar, 2000). Although, phenol has significant inhibitory effects on
growth of microorganism at higher concentrations (Kumar and
Kumar, 2005), the isolate A235 was gradually adapted simulta-
neously to increased concentrations of phenol and lower concen-
tration of yeast extract in the growth medium. As shown in Fig. 1,
phenol biodegradation by the adapted cells was two times higher
than for the non-adapted ones. In a similar study, Kwon and Yeom
(2009) indicated that pre-adaptation enhanced the phenol degra-
dation rate of Pseudomonas fluorescence KNU417 by almost two
times. In the current study, in addition to phenol adaptation we also
develop toluene-adapted cells. The toluene-adapted cultures
degraded phenol significantly better than the phenol-adapted cells
(Fig. 1). A previous report also showed that benzene- or toluene-
adapted cultures degraded phenol more efficiently than the
phenol-adapted ones (Yeom et al., 1997).
3.3. Phenol degradation and cell growth
The profiles of growth and of phenol degradation by isolate
A235 at different initial phenol concentrations (50e200 mg l
1) are
showed in Fig. 2. At the initial phenol concentration of 50 mg l
1, no
lag phase was observed. However, when phenol was used as a sole
substrate at initial concentrations of 75, 100, 125, 150 and
200 mg l
1, lag phases of respectively 48 h, 72 h, 98 h, 144 h and
240 h were observed (Fig. 2A). The observation that the lag phase
increases when phenol concentration is increased has also been
reported previously (Kumar and Kumar, 2005; Agarry et al., 2008).
Our data clearly show that, for all tested concentrations, the
isolate A235 degraded between 20 and 80% of phenol within 15
days. The rate of degradation was reduced when the initial phenol
concentration was increased, probably due to a substrate inhibition
effects (Fig. 2B). Agarry et al. (2008) reported a similar observation
for a Pseudomonas strain grown on phenol. Peyton et al. (2002) also
observed that the specific growth rates decreased as the initial
phenol concentration increased in high salt solutions. These results
suggest that phenol is an inhibitory substrate for halophilic
microorganisms.
The growth kinetics of isolate A235 at phenol concentrations
between 50 and 200 mg l
1 has been determined using the Monod
model. The Monod parameters values were respectively 0.015 h
1
for the mmax and 71.4 mg l
1 for the Ks. The observed specific
growth rates showed an increasing tendency up to 200 mg l
1.
120
Phenol concentration (mg l )
100
80 T oluene Adaptation
60 Phenol adaptation
40 No adaptation
20
0
0 100 200 300 400
Time (h)
Fig. 1. Biodegradation of phenol by toluene and phenol adapted A235 strain.
 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146 143

1.6 0.8

1.4

Optical density (600 nm)

Optical density (600 nm)

1.2 0.6

1
0.4
0.8

0.6
0.2
0.4
0.2
0
0 0 50 100 150 200 250 300 350 400
0 100 200 300 400
Time (h)
Time (h) A
A
120

Phenol concentration (mg l )

-1
250
100
Phenol concentration (mg l )
-1

200 80

60
150
40

20
100
0
50 0 50 100 150 200 250 300 350 400

0 B
0 100 200 300
Time (h)
B Fig. 3. Effects of inoculum size on (A) growth and (B) phenol degradation capacity of
Fig. 2. Cell growth (A) and phenol degradation (B) capacity of A235 isolate at various
initial concentrations of phenol. Concentrations (mg/l) were represented as follows:
(filled diamond) 50, (open square) 75, (filled triangle) 100, (open triangle) 125; (filled
square) 150, (open circle) 200.
Studies on the growth kinetics of archaea are limited and the cur-
rent study is the first one reporting phenol degradation by a
halophilic archaeal strain. Christen et al. (2011) reported that Sul-
folobus solfataricus 98/2, a thermoacidophilic archaeon, acclima-
tized to phenol on a mixture of glucose and phenol showed a
specific growth rate value of 0.034 h
1 at phenol concentrations
lower than 365 mg l
1. Furthermore, the Ks value was 77.7 mg l
1
for the cells of S. solfataricus 98/2 acclimatized on 51e745 mg l
1 of
phenol. While most of the Ks values found for non-halophilic mi-
croorganisms ranged from 1 to 110 mg l
1 (Onysko et al., 2000;
Kumar and Kumar, 2005), we found Ks as 71.4 mg l
1 in the cur-
rent study.
3.4. Optimization of phenol biodegradation
The effects of the inoculum size on the rate of phenol degra-
dation and biomass production were examined for cultures grown
in the presence of 100 mg l
1 phenol. The phenol biodegradation
rate as well as the biomass increased as the inoculum size increased
whereas, the lag phase dropped (Fig. 3A). Haddadi and Shavandi
(2013) also found a similar relationship between the inoculum
size and the rate of phenol biodegradation and biomass production
for a Halomonas sp. In their study, the optimal inoculum size was
found to be 10% corresponding to approximately 80% of phenol
degradation. In our study, the phenol degradation was increased by
about 30% at optimal inoculum size (Fig. 3B). A similar result has
been reported for the strain SM5 grown on phenol where the
optimal inoculum size was 12.5% (Hong-Xia, 2011). Santos and
Linardi (2004) noticed that the starting inoculum was a very
important parameter when cultivation was carried out in media
Time (h)
400
A235 strain at 100 mg/l phenol (filled diamond, 2.5%; open square, 5%; filled triangle,
10%; asterisk, 15%).
containing toxic compounds, like phenol. In another study (Beshay
et al., 2002), the rate of phenol biodegradation was significantly
affected by the inoculum size; by increasing it, the lag phase
decreased and the rate of phenol biodegradation was enhanced.
It has been shown that several neutral or alkaliphilic microor-
ganisms such as Arhodomonas aquaeolei, Modicisalibacter tuni-
siensis, Penicillium chrysogenum, Candida tropicalis and Alcaligenes
faecalis have the ability of degrading phenols at various NaCl con-
centrations (ranging from 0.25 to 15%) (Hinteregger and
Streichsbier, 1997; Bastos et al., 2000; Alva and Peyton, 2003;
Bonfa et al., 2013; Haddadi and Shavandi, 2013). Optimal biodeg-
radation of phenol by a moderately halophilic bacterial consortium
was performed in the presence of 5% (w/v) NaCl (Veenagayathri
and Vasudevan, 2011). However, at higher NaCl concentrations,
longer lag periods were observed and longer times were required
for complete phenol degradation which required respectively 50 h
and 100 h a 9% and 14% (w/v) NaCl (Hinteregger and Streichsbier,
1997).
In the current study, we investigated the effects of various NaCl
concentrations on the growth of isolate A235 and on its ability to
degrade phenol. We found that it could degrade phenol at high
NaCl concentrations. The highest degradation of phenol (over 80%)
and shortest lag phase were obtained in cultures containing 20%
(w/v) NaCl (Fig. 4). However, the degradation capacity of A235
decreased to 40% when NaCl concentration was increased to 25%
(w/v). At this high concentration, the growth rate was also
decreased; suggesting extreme salinity might become a natural
barrier to hydrocarbon metabolism as suggested previously by
Ward and Brock (1978).
At the end of the 15th day of incubation, the highest phenol
degradation, associated with the highest growth of the isolate, was
obtained at neutral pH (7.5). It is well documented that the majority
of halophilic archaea grow at neutral pH (Bowers and Wiegel,
144 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146

0.9 0.9

Optical density (600 nm)

Optical density (600 nm)

0.7
0.7

0.5
0.5

0.3
0.3 0 100 200 300 400
0 100 200 300 400
Time (h) Time (h)
A
A

120
120
Phenol concentration (mg l )
-1

100

Phenol concentration (mg l )

-1
100
80
80
60
60
40
40
20
20
0
0 100 200 300 400 0
B 0 100 200 300 400
Time (h)
Time (h)
Fig. 4. Effects of NaCl concentrations on (A) growth and (B) phenol degradation ca- B
pacity of A235 strain at 100 mg/l phenol (open triangle, 5%; open square, 10%; filled
triangle, 15%; filled square, 20%; filled diamond, 25%). Fig. 5. Effects of yeast extract on (A) growth and (B) phenol degradation of A235 strain
(open circle 0.3%; filled square, 0.1%; filled triangle, 0.05%; open square, 0.025%; filled
diamond, none).
2011). In this study, we also found out that the pH values lower or
higher than 7.5 significantly affected phenol biodegradation effi-
increased slightly after the addition of 0.025% yeast extract and
ciency. The pH conditions for moderately halophilic microorgan-
thereafter the degradation rate decreased with the increased con-
isms which have the ability to degrade phenol were observed to be
centration of yeast extract. While the increasing concentration of
in the range of pH 7.0 to 9.5 (Peyton et al., 2002; Alva and Peyton,
yeast extract significantly stimulated growth, phenol degradation
2003; Hong-Xia, 2011; Veenagayathri and Vasudevan, 2011).
was repressed. This might be due to the fact that yeast extract acts
The isolate A235 was able to grow and degrade phenol at tem-
as a competing growth substrate. Several other researchers also
peratures ranging from 25 to 50  C. However, the greatest growth
reported that the addition of yeast extract in the medium enhanced
and phenol degradation were observed at 37  C. Hong-Xia, (2011)
the removal efficiency of phenol by microorganisms (Woolard and
showed that phenol degradation was most efficient at 37  C for
Irvine, 1994; Veenagayathri and Vasudevan, 2011). It is well
the marine bacterial strain SM5. The results suggest that the tem-
established that complex nutrients, particularly nitrogen, are
perature is critical after 37  C, and a further increase in temperature
required for the stimulation of cell growth under high salt condi-
may hinder phenol degradation. El-Naas et al. (2009) suggested
tions (Oren et al., 1992). Nicholson and Fathepure (2004) reported
that high temperature may have adverse effects on the enzymes
the degradation of the benzene-toluene-ethylbenzene-xylene
that are responsible for the aromatic ring cleavage. Similarly, our
(BTEX) mixture by halophilic and halotolerant bacteria. They
data show that temperature affects the phenol degrading ability of
observed the complete degradation of benzene in the presence of
the archaeal isolate A235.
yeast extract, vitamins or trace elements.
A series of experiments were performed to establish the effect of
agitation on phenol degradation. Of all the measured variables, the
lowest rate of phenol degradation was observed under static con- 3.5. The phenol metabolic pathway of A235 isolate
dition. This was most likely caused by poor oxygenation. Higher
phenol degradation rates were obtained when the agitation was The identification of products formed during the biodegradation
increased. The optimal degradation was obtained when cultures process of phenol is essential for a better understanding of the
were grown at 200 rpm. Similarly, Oboirien et al. (2005) reported degradation mechanisms. Phenol is aerobically converted to cate-
that the biodegradation rate increases with elevated aeration rates. chol and subsequently, the catechol is degraded via the ortho or
The results showed that the rate of agitation plays very significant meta fission pathway to intermediates of central metabolism. The
role on the degradation of phenol. It is well known that oxygen ring cleavage of catechol is catalyzed by the ortho cleaving enzyme,
solubility decreases under high salinity. Therefore, it is vital to catechol 1,2-dioxygenase or by the meta cleaving enzyme, catechol
allow more oxygen to be dissolved under high salinity conditions. 2,3-dioxygenase (Gurujeyalakshmi and Oriel, 1989). The metabo-
The effect of yeast extract on phenol degradation and growth of lites formed during phenol degradation were identified by using
isolate A235 are presented in Fig. 5. Phenol removal efficiency Rothera test as described in the Materials and Methods section.
 E. Acikgoz, B. Ozcan / International Biodeterioration & Biodegradation 107 (2016) 140e146 145

Data show that isolate A235 possesses the meta pathway, because
the development of a yellow product was observed which indicates
the presence of 2-hydroxymuconic semialdehyde, the product of
catechol 2,3-dioxygenase (Li and Humphrey, 1989). To confirm the
results, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase
activities were assayed in cell-free extracts. Absence of absor-
bance at 260 nm indicated the absence of the ortho-cleavage
pathway. However, absorbance at 375 nm, which is due to formation
of 2-hydroxymuconic semialdehyde, confirmed the meta-cleavage
pathway. Therefore, it is likely that strain A235 metabolizes phenol
through the meta-cleavage pathway.
3.6. Effects of salt concentrations, temperatures and pH on catechol
2,3-dioxygenase activity
The effects of salt concentration on the catechol 2,3-
dioxygenase activity of the strain A235 was determined for the
salt concentrations ranging from 1.5 to 3.5 M KCl and NaCl. The
activity of the enzyme was more strongly stimulated by KCl than by
NaCl. The maximal activity was obtained at 2 M KCl (Fig. 6A). KCl
plays an important role in the cell by maintaining the enzymes in
an active state (Oren and Mana, 2002). Ortega et al. (2011) also
reported that the intracellular enzyme from Haloferax volcanii was
more active at higher concentrations of KCl when compared with
NaCl. It is also established that the [Naþ] is usually lower inside the
cells than outside, while the [Cl
] remains approximately equal to
the external concentration, and [Kþ] is higher inside than outside
the cells (Grant, 2004).
The maximal catechol 2,3-dioxygenase activity occurred at 42  C
and at 2 M KCl. (Fig. 6B). Catechol 2,3-dioxygenase activity isolated
from non-halophilic organisms was reported for Pseudomonas sp.
ZJF08, Planococcus sp. strain S5, having activity at 30e70  C (Zhou
et al., 2007; Hupert-Kocurek et al., 2012).
The influence of pH on the catechol 2,3-dioxygenase activity of
the isolate A235 was determined at 42  C in the culture containing
2 M KCl using catechol as a substrate (Fig. 6C). The optimal pH was
determined to be pH 8.0. The optimal pH for the catechol 2,3-
dioxygenase from Planococcus sp. S5 was also found to be pH 8.0
(Hupert-Kocurek et al., 2012). The optimal pH for catechol 2,3-
dioxygenase for the thermo-acidophilic archaeon S. solfataricus
strain 98/2 was found to be lower (pH 7.0e7.5) (Murakami et al.,
1998).
Acknowledgements

This study was supported by the Research Project Units of

8 Mustafa Kemal University (project number 1105Y0106).
Activity (U ml )
-1

6
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